A tubular microdialysis probe is made from polysulfone hollow fibre for human haemodialysis, which has an inner diameter of 200 μm and a thickness of 20 μm. Milk is deposited to the outer surface of the hollow fibre and allowed to dry to form a dry sample. The tubular probe is then connected to the syringe pump and microdialysis of the dry sample into 0.5 mol/L HCl as acceptor is performed. 2.5 μL of microdialysate is obtained and analyzed for inorganic cations by capillary electrophoresis with contactless conductivity detection. Baseline separation of NH4+, K+, Ca2+, Na+, Mg2+, Li+ is achieved in 5.5 mol/L acetic acid as background electrolyte using a fused silica capillary with inner diameter of 25 μm and length of 31.5 cm. The reproducibility of dry sample microdialysis including CE analysis for peak area ranges from 2.4 to 3.9 % after normalization to Li+ as internal standard.
- MeSH
- elektroforéza kapilární * přístrojové vybavení metody MeSH
- kationty * analýza MeSH
- mikrodialýza * přístrojové vybavení metody MeSH
- mléko * chemie MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
BACKGROUND: The availability of dialysis membranes in the form of hollow fibres with diameters compatible with the fused silica capillaries used in capillary electrophoresis is very limited. However, haemodialysis bicarbonate cartridges commonly used in human medicine containing polysulfone hollow fibres are available on the market and are used for the fabrication of coaxial microdialysis probes. The miniature probe design ensures that steady-state conditions are achieved during microdialysis of minimal volumes of body fluids. RESULTS: A coaxial microdialysis probe with a length of 5 cm and an inner diameter of 200 μm is used for microdialysis of 10 μL of body fluid collected into a sampling fused silica capillary with an inner diameter 430 μm. Microdialysis is performed into 0.01 M HCl as a perfusate at stopped flow and 2 μL of the resulting microdialysate are subjected to analysis by capillary electrophoresis with contactless conductivity detection. Microdialysis pre-treatment is verified by analysis of 11 common amino acids at a 100 μM concentration level, resulting in recoveries of 98.3-102.5%. The electrophoretic separation of amino acids is performed in 8.5 M acetic acid at pH 1.37 as a background electrolyte with analysis time up to 4.5 min and LOD in the range of 0.12-0.28 μM. The reproducibility of the developed technique determined for the peak area ranges from 1.2 to 4.5%. Applicability is tested in the quantification of valine and leucine in plasma during fasting and subsequent reconvalescence. SIGNIFICANCE: The fabrication of a coaxial microdialysis probe for the laboratory preparation of microliter volumes of various types of clinical samples is described, which is coupled off-line with capillary electrophoretic monitoring of amino acids in 2 μL volumes of microdialysate. The developed methodology is suitable for quantification of 20 amino acids in whole human blood, plasma, tears and has potential for analysis of dry blood spots captured on hollow fibre.
- MeSH
- aminokyseliny * MeSH
- elektroforéza kapilární MeSH
- lidé MeSH
- mikrodialýza MeSH
- oxid křemičitý MeSH
- reprodukovatelnost výsledků MeSH
- tělesné tekutiny * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The review presents an evaluation of the development of on-line, at-line and in-line sample treatment coupled with capillary and microchip electrophoresis over the last 10 years. In the first part, it describes different types of flow-gating interfaces (FGI) such as cross-FGI, coaxial-FGI, sheet-flow-FGI, and air-assisted-FGI and their fabrication using molding into polydimethylsiloxane and commercially available fittings. The second part deals with the coupling of capillary and microchip electrophoresis with microdialysis, solid-phase, liquid-phase, and membrane based extraction techniques. It mainly focuses on modern techniques such as extraction across supported liquid membrane, electroextraction, single drop microextraction, head space microextraction, and microdialysis with high spatial and temporal resolution. Finally, the design of sequential electrophoretic analysers and fabrication of SPE microcartridges with monolithic and molecularly imprinted polymeric sorbents are discussed. Applications include the monitoring of metabolites, neurotransmitters, peptides and proteins in body fluids and tissues to study processes in living organisms, as well as the monitoring of nutrients, minerals and waste compounds in food, natural and wastewater.
- MeSH
- elektroforéza kapilární metody MeSH
- elektroforéza mikročipová * metody MeSH
- mikrodialýza MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Sensitive electrophoretic determination of 3-hydroxybutyrate (3HB) as an indicator of human ketogenesis is performed in fused silica capillary covalently coated by an anionic copolymer of poly(acrylamide-co-sodium-2-acrylamido-2-methylpropanesulphonate) (PAMAMPS). Baseline separation of 3HB from other components of human serum is achieved in a 20 μm capillary with an effective length of 17 cm covered by 4% PAMAMPS, which generates a cathodic EOF with a mobility of 8.30 ± 0.00 · 10-9 m2/V.s in 80 mM MES/His as background electrolyte. 3HB migrates in counter-current electrophoretic mode against EOF, that effectively improving electrophoretic resolution. Sample pre-treatment is based on adding of 45 μL acetonitrile to 15 μL serum and, after shaking, a 28 mm long zone of supernatant is injected into the capillary, and sharpened after turning on a separation voltage of 20 kV using the technique of large volume sample stacking, where the EOF forces the residual acetonitrile from the capillary. When combined with universal contactless conductivity detection, the achieved LOD and LOQ are 0.43 μM and 1.44 μM, respectively, that are sufficiently low for monitoring the physiological 3HB level. The performed clinical study subsequently showed that serum 3HB increases from a concentration of 71 μM, corresponding to normal food, to level of 1924 μM after 60 h of fasting and returns to the normal physiological concentration 48 h after commencing consumption of high-saccharide food.
- MeSH
- acetonitrily MeSH
- akrylové pryskyřice MeSH
- alkylsulfonany MeSH
- elektroforéza kapilární * metody MeSH
- kyselina 3-hydroxymáselná MeSH
- lidé MeSH
- omezení příjmu potravy * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Determination of the broad-spectrum antibiotics amoxicilline (AMX) and ceftazidime (CTZ) in blood serum and microdialysates of the subcutaneous tissue of the lower limbs is performed using CE with contactless conductivity detection (C4 D). Baseline separation of AMX is achieved in 0.5 M acetic acid as the background electrolyte and separation of CTZ in 3.2 M acetic acid with addition of 13% v/v methanol. The CE-C4 D determination is performed in a 25 μm capillary with suppression of the EOF using INST-coating on an effective length of 18 cm and the attained migration time is 4.2 min for AMX and 4.4 min for CTZ. The analysis was performed using 20 μl of serum and 15 μl of microdialysate, treated by the addition of acetonitrile in a ratio of 1/3 v/v and the sample is injected into the capillary using the large volume sample stacking technique. The LOQ attained in the microdialysate is 148 ng/ml for AMX and 339 ng/ml for CTZ, and in serum 143 ng/ml for AMX and 318 ng/ml for CTZ. The CE-C4 D method is employed for monitoring the passage of AMX and CTZ from the blood circulatory system into the subcutaneous tissue at the sites of diabetic ulceration in patients suffering from diabetic foot syndrome and also for measuring the pharmacokinetics following intravenous application of bolus antibiotic doses.
This review article summarises aspects of the determination of amino acids using capillary and chip electrophoresis in combination with contactless conductivity detection from their historical beginnings to the present time. Discussion is included of the theory of conductivity detection in electromigration techniques, the design of contactless conductivity cells for detection in capillaries and on microchips, including the use of computer programs for simulation of the conductivity response and the process of the electrophoretic separation of amino acids. Emphasis is placed on optimisation of the background electrolyte composition, chiral separation, multidimensional separation, stacking techniques and the use of multidetection systems. There is also a description of clinical applications, the determination of amino acids in foodstuffs, waters, soils and composts with emphasis on modern techniques of sample treatment, such as microdialysis, liquid membrane extraction and many other techniques.
Electrophoretic stacking is developed for sensitive determination of three zwitterionic antiepileptics, namely vigabatrin, pregabalin and gabapentin, in human serum. CE separation is performed in a 25 μm fused silica capillary covalently coated with the copolymer of acrylamide with 5% content of permanently charged 3-acrylamidopropyl trimethylammonium chloride (PAMAPTAC). In background electrolyte of 500 mM acetic acid, the 5% PAMAPTAC generates an anodic electro-osmotic flow with a magnitude of (-18.6 ± 0.5) · 10-9 m2V-1s-1, which acts against the direction of the electrophoretic migration of the analytes. A sample of the antiepileptic prepared in a 25% v/v infusion solution and 75% v/v acetonitrile is injected into the capillary in a large volume attaining a zone length of up to 270 mm. After turning on the separation voltage, the antiepileptics are isotachophoretically focussed behind the zone of Na+ ions with a sensitivity enhancement factor of 78. For the clinical determination of antiepileptics, the human serum is diluted with acetonitrile in a ratio of 1:3 v/v and a zone with a length of 90 mm is injected into the capillary. The method is linear in the 0.025-2.5 μg/mL concentration range; the attained limit of quantification is in the range 18.3-22.8 nmol/L; the within-day precision for the migration time is 0.8-1.2% and for the peak area 1.5-2.4%.
- MeSH
- antikonvulziva * MeSH
- chloridy * MeSH
- elektroforéza kapilární MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Branched chain amino acids (BCAAs), alanine and glutamine are determined in human plasma by capillary electrophoresis with contactless conductivity detection (CE/C4 D). The baseline separation of five amino acids from other plasma components is achieved on the short capillary effective length of 18 cm in 3.2 mol/L acetic acid with addition of 13% v/v methanol as background electrolyte. Migration times range from 2.01 min for valine to 2.84 min for glutamine, and LODs for untreated plasma are in the interval 0.7-0.9 μmol/L. Sample treatment is based on the addition of acetonitrile to only 15 μL of plasma and supernatant is directly subjected to CE/C4 D. Circulating amino acids are measured in patients with pancreatic cancer and cancer cachexia during oral glucose tolerance test. It is shown that patients with pancreatic cancer and cancer cachexia syndrome exhibit low basal circulating BCAAs and glutamine levels and loss of their insulin-dependent suppression.
Asymmetric and symmetric dimethylarginines are toxic non-coded amino acids. They are formed by post-translational modifications and play multifunctional roles in some human diseases. Their determination in human blood plasma is performed using capillary electrophoresis with contactless conductivity detection. The separations are performed in a capillary covered with covalently bonded PAMAPTAC polymer, which generates anionic electroosmotic flow and the separation takes place in the counter-current regime. The background electrolyte is a 750 mM aqueous solution of acetic acid with pH 2.45. The plasma samples for analysis are treated by the addition of acetonitrile and injected into the capillary in a large volume, reaching 94.5% of the total volume of the capillary, and subsequently subjected to electrophoretic stacking. The attained LODs are 16 nm for ADMA and 22 nM for SDMA. The electrophoretic resolution of both isomers has a value of 5.3. The developed method is sufficiently sensitive for the determination of plasmatic levels of ADMA and SDMA. The determination does not require derivatization and the individual steps in the electrophoretic stacking are fully automated. The determined plasmatic levels for healthy individuals vary in the range 0.36-0.62 µM for ADMA and 0.32-0.70 µM for SDMA.
Capillary electrophoretic separation of ketamine, norketamine, hydroxynorketamine, and dehydronorketamine was performed in the counter-current regime under the influence of oppositely-directed electroosmotic flow. For this purpose, the fused silica capillaries were covalently coated with the poly(acrylamide-co-3-acrylamidopropyl trimethylammonium chloride) copolymer (PAMAPTAC). The content of the cationic monomer APTAC in the polymerization mixture varied in the range 0-6 mol. % and the generated electroosmotic flow increased continuously in the 0-20 · 10-9 m2V-1s-1 interval. Importantly, it resulted in improved electrophoretic resolution of ketamine/norketamine, which increased from 0.8 for neutral PAM coating (i.e. 0% PAMAPTAC) to 3.0 for 6% PAMAPTAC. The determination of ketamine and its derivates in rat serum was performed in a 4% PAMAPTAC capillary with an inner diameter of 25 μm. The separation was performed in a 500 mM aqueous solution of acetic acid (pH 2.3). The clinical sample was deproteinized by the addition of acetonitrile to the serum and a large volume of the treated sample was injected directly into the capillary. The achieved limit of detection ranged from 2.2 ng/mL for dehydronorketamine to 4.1 ng/mL for hydroxynorketamine; the intra-day repeatability was 1.0-1.5% for the migration time and 2.8-3.3% for the peak area. The developed methodology was employed for time monitoring of ketamines in rat serum after intra venous administration of low doses of anaesthetic at a level of 2 μg per g of body weight.
- Publikační typ
- časopisecké články MeSH
